Increased Mitogen-Activated Protein Kinase Activity and TNF-α Production Associated with Mycobacterium smegmatis- but Not Mycobacterium avium-Infected Macrophages Requires Prolonged Stimulation of the Calmodulin/Calmodulin Kinase and Cyclic AMP/Protein Kinase A Pathways

ERK1/2 activation but not p38 activation requires intracellular Ca²⁺ upon mycobacterial infection. Shown are the Western blots of lysates probed for activated ERK1/2 and p38 after infection with M. smegmatis or M. avium 724 in BMMφ. Macrophages were treated with BAPTA-AM or DMSO, as a vehicle control (−), at the time of infection. One-hour postinfection, the cell lysates were analyzed by Western blotting using phospho-specific Abs to ERK1/2 and p38 as described in Materials and Methods. Blots were then probed with total ERK1/2 Ab to show equal protein loading. These results are representative of three separate experiments.

CaMKII is activated in macrophages infected with mycobacteria and is required for early and sustained activation of ERK1/2 in mycobacterial-infected BMMφ. A, BMMφ were infected with M. smegmatis or M. avium 724 for 15 min, 6 h, and 9 h as described in Materials and Methods. Cell lysates were removed and screened for CaMKII activity. Values are expressed as mean ± SD. ∗, Significant to RC. ∗∗, Significant to RC and M. avium 724 (p < 0.05). Similar results were observed in three separate experiments. B, Dose-dependent inhibition of ERK1/2 was seen with varying concentrations of KN-62 in BMMφ infected with M. smegmatis for 1 h. C–E, BMMφ were treated with W-7, KN-62, KN-93, or DMSO (−) as vehicle control, for 30 min before the infection with M. smegmatis or M. avium 724 for 1 h (C and E) and 9 h (D and F) and lysed after the indicated times. For the 9-h infection, cells were infected with mycobacteria for 4 h and washed, and fresh medium containing DMSO or inhibitors was added, and the cells were harvested after an additional 5 h. BMMφ cell lysates were probed for activated ERK1/2 and p38. Total ERK1/2 blots were run to show equal protein loading. Results are representative of three separate experiments.

M. smegmatis-infected BMMφ produce significantly higher levels of cAMP as compared with M. avium-infected BMMφ. Macrophages were infected with M. avium 724 or M. smegmatis for 10 min, 30 min, 1 h, 2 h, 6 h, or 8 h. Cell lysates were prepared for each time point and used for subsequent cAMP ELISA (A). cAMP production in BMMφ after a 30-min infection (B) and a 6-h infection (C). ∗, p < 0.05 by two-tailed Student’s t test. §, M. smegmatis is significant to RC at p < 0.05 by two-tailed Student’s t test. Values are expressed as mean ± SD. These data are representative of three separate experiments.

Dose response of ERK1/2 activation in BMMφ infected with M. smegmatis for 1 h following H89 treatment. Shown are Western blots of BMMφ cell lysates probed for activated ERK1/2 after infection with M. smegmatis. Macrophages were treated with 5, 10, 20, 30, and 50 μM H89 or DMSO vehicle control (−) for 1 h before a 1-h infection with M. smegmatis. The relative densities of the upper ERK1/2 band were analyzed by densitometry. AU, Arbitrary units. The results are representative of two separate experiments.

ERK1/2 activation at 1- and 9-h postinfection requires PKA activity in both M. smegmatis- and M. avium-infected BMMφ. Shown are Western blots of BMMφ cell lysates probed for activated ERK1/2 after infection with M. smegmatis or M. avium 724 for 1 h (A) and 9 h (B) and for 1 h in the KT5720 experiment (C). Macrophages were treated with H89, KT5720, or DMSO (−), 1 h before infection and lysed after indicated times. For the 9-h infection, cells were treated the same way as described in Fig. 3.

Increased cAMP activation in BMMφ following M. smegmatis infection is dependent on the CaM/CaMK pathway. BMMφ were treated with KN-62 or W7 or with DMSO, as a vehicle control, for 30 min before infection as described in Fig. 3. Macrophages were infected with M. smegmatis or M. avium 724 and harvested at the indicated times, and cell lysates were analyzed for cAMP concentration by ELISA. Values are expressed as mean ± SD. These data are representative of three separate experiments.

TNF-α production in BMMφ is dependent on the CaM/CaMK and cAMP/PKA pathways following an M. smegmatis but not M. avium 724 infection. BMMφ cells were treated with KN-62, W7, or DMSO for 30 min (A) and with H89 or DMSO for 1 h (B), before the addition of mycobacteria. Infection was continued for a total of 9 h, as described in Fig. 3. Culture supernatants were analyzed for TNF-α by ELISA. Values are expressed as mean ± SD. The results are representative of three separate experiments.